Intracellular pathogens remain a major cause of global mortality and morbidity. By evasion of extracellular defenses, such as antibodies, intracellular pathogens pose a unique challenge to the immune systems of their hosts. A fundamental question we address is how intracellular pathogens are sensed by the innate immune system. In the past few years, we and others have discovered that intracellular pathogens trigger a variety of different cytosolic immunosurveillance pathways. These pathways appear to play important roles in shaping protective innate and adaptive responses. However, the molecular basis of cytosolic immunosurveillance remains poorly characterized, and it is likely that additional pathways remain to be discovered. Our studies are motivated by our belief that a better understanding of innate immunity is central to the improved design of new vaccines, adjuvants and immunotherapeutics. Exciting recent work from our group and others has shown that unique bacterial nucleotides, cyclic-di-GMP and cyclic-di-AMP, are novel and potent stimulator of innate and adaptive immune responses. The field now confronts a major outstanding question: what is the host sensor that specifically detects cyclic-di-nucleotides? We are taking genetic and biochemical approaches to address this fundamental question. In what we believe is an exciting advance, our unpublished experiments have now converged on identification of the sensor as a host protein called Sting (Stimulator of interferon genes). We identified a novel Sting mutant mouse in a forward genetic screen using ENU mutagenesis in mice. Using this mutant, we found Sting is essential for responses to c-dinucleotides. In addition, we have recently found that radiolabelled c-di-GMP binds specifically to Sting. Thus, our Specific Aims are: (1) Determine how the interaction of Sting with cyclic-di-nucleotides leads to an IFN response (2) Distinguish the role of Sting in the cytosolic response to cyclic-di-nucleotides from the role of Sting in the cytosolic response to DNA, and distinguish these functions of Sting in the response to three intracellular bacterial pathogens (L. monocytogenes, M. tuberculosis, and L. pneumophila) (3) Identify novel regulators of innate immunity to three intracellular bacterial pathogens (L. monocytogenes, M. tuberculosis, and L. pneumophila) by continuing to perform forward genetic screens in mice.